Nanofluidic DNA separation media promise to reduce the time required to resolve long DNA (> 20 kbp) by size from days to minutes. Amongst these new media, the most promising and well-developed are artificial gels formed by arrays of cylindrical posts. However, the translation of nanofluidic devices from the proof-of-principle stage into routine use in biology and medicine has been hampered by an absence of engineering analyses that relate the device design to its performance. The long-term goal of this research program is to develop, from a systematic and quantitative basis, DNA electrophoresis devices whose geometries are specifically designed to separate a given DNA size-range. The objective of this particular application is to engineer nanopost arrays for sep- arating long DNA. To accomplish this goal, Brownian dynamics simulations will be used to design the devices, single-molecule videomicroscopy will be used to validate the simulations, and analytical separation experiments will be used to evaluate the device performance. Preliminary results, based on this approach, contradict the con- ventional wisdom that the posts need to be closely spaced or randomly distributed to ensure frequent collisions with the DNA. Rather, these preliminary data indicate that sparse ordered arrays have the potential to greatly increase the separation power of nanopost separation media. Based upon this insight, the research is plan is divided into four specific aims: Specific Aim 1: Demonstrate, via experiments, long DNA separations in a sparse hexagonal nanopost array; Specific Aim 2: Develop a quantitative simulation model and validate it with the latter experiments; Specific Aim 3: Use this simulation model and further experiments to evaluate a non-hexagonal array geometry; Specific Aim 4: Engineer the most promising geometry to size a relevant genomic sample. This research is significant because it will lead to improvements in the analysis of genomes. Although second- generation sequencers may ultimately prove to be the most suitable method for re-sequencing projects, DNA fingerprinting and physical mapping will remain important for the assembly of unknown genomes and low- cost screening applications. The devices produced by this research will impact genome analysis by providing optimized systems for sizing DNA in the range relevant for restriction digests of BAC clones. This work is innovative because it counters the current trend in nanofluidic devices towards dense arrays for DNA separations. The research takes advantage of a synergistic combination of focused experimentation and systematic simulation studies, a design approach which has not been employed thus far. Taken as a whole, this research program will impact the larger field of biomedical microfluidics and nanofluidics by providing a systematic engineering framework for translating DNA electrophoresis devices from the proof-of-principle stage to optimized devices. PUBLIC HEALTH RELEVANCE: The proposed work will lead to improved nanoscale systems for rapid and high-resolution separations of long DNA. These devices will accelerate a number of key genomics applications, such as DNA fingerprinting of infec- tious organisms and genome assembly.